The invention relates to the field of cancer diagnosis and the application of diagnostic techniques in pathology and haematology. Specifically, the invention relates to techniques that indicate presence of chromosomal aberrations by detecting tumour-specific gene products that are exclusively expressed by tumour cells containing said chromosomes.
Chromosomal abnormalities or aberrations are a leading cause of genetic disorders or diseases, including congenital disorders and acquired diseases, such as malignancies. Malignant cells have a common clonal origin as they are supposed to originate from a single autonomously growing cell that withdrew from environmental growth regulating signals.
The term xe2x80x98cancerxe2x80x99 comprises a heterogeneous group of neoplasms, in which each type has its own characteristic when considering its malignant potential and its response to therapy. Currently, the effectiveness of cancer treatment is empirically determined. Depending on the moment in time in the development of cancer, the origin and spread of the cancer and on the physiological condition of the patient, the most proper and most effective treatment is selected. At present, selections from surgical treatment, radiation therapy and chemotherapy (or combinations of the former therapies) can be made. Yet, it is realised that each therapy bears side-effects that compromise the benefits of treatment enormously. It goes without saying that accurate diagnosis of the various cancer types is pre-eminent in helping select the most effective therapy.
The basis of cancer stems from chromosomal aberrations such as translocations, inversions, insertions, deletions and other mutations within or among chromosomes. Often, one chromosome or two different chromosomes are involved in the development of malignancies. In this way, genes or fragments of genes are removed from the normal physiological context of the non-aberrant chromosome and fuse with or find a location in a recipient chromosome, (be it the same or a second chromosome) adjacent to non-related genes or fragments of genes (often oncogenes or proto-oncogenes), where the new genetic combination can be the foundation of a malignancy.
Rearrangements, such as translocations happen often in a somewhat established pattern, where genes, or fragments thereof, are removed from the non-aberrant chromosome at a breakpoint or breakpoint cluster region, and are inserted in the recipient chromosome at a fusion region, thereby creating rearranged, deleted, translocated or fused genes that are specific for that specific cancer. Moreover, rearrangements or translocations can be reciprocal, in that two chromosomes exchange parts which leads to cells containing two, reciprocally rearranged chromosomes which both contain new fused genes.
When the fused gene is translated it generates a gene-product, mRNA, that is unique for the tumour. The chimeric mRNA comprises parts or fragments of two mRNA""s that correspond to and were originally transcribed by the originally separated genes. This tumour-specific mRNA is uniquely characterised by a fusion point, where the RNA fragments meet. In some cases, these fusion points can be detected by hybridising nucleic acid probes. However, considering the large variation within the individual rearrangements seen in these translocations and depending on the localisation of the breakpoint within the non-aberrant gene whereby (even when the translocations occur within the same two genes) different tumour-specific genes can be generated, it is deemed likely that within each separate case of these types of cancer, new fusion points arise. Detection of cancer by specific detection of the fusion-point of the tumour-specific gene-product (mRNA) has therefore never been widely applicable.
When the fused gene is fused in frame, the fused mRNA is translated into a fusion protein that is unique for the tumour. The protein comprises parts of two proteins that correspond to and were originally transcribed by and translated from the originally separated genes. Tumour-specific proteins are uniquely characterised by a fusion point, where the two proteins meet. Fusion points are antigenically exposed, comprising distinct epitopes which sometimes can be immunologically detected. However, considering the large variation within the individual rearrangements seen in these translocations and depending on the localisation of the breakpoint within the non-aberrant gene whereby (even when the translocations occur within the same two genes) different tumour-specific genes can be generated, it is deemed likely that within each separate case of these types of cancer, new fusion points arise. Detection of cancer by specific detection of the fusion-point epitope of the tumour specific protein has therefore never been widely applicable. The tumour-specific gene products (fusion products) of the fused or rearranged genes may contribute to the further development of the cancer.
An area where chromosome aberrations are relatively well studied (as compared with other cancer types) is the field of leukaemia. Comparable to most malignant tumours, leukaemia""s differ in the degree of differentiation of tumour cells. According to clinical presentation, leukaemia""s are divided in acute and chronic forms, depending on the rapidity with which they evolve and, if untreated, cause death.
Depending on the cell lineage(s) involved in the leukaemic process, acute leukaemias are classified as acute lymphoblastic leukaemias (ALL) and acute non-lymphoblastic leukaemias (ANLL), with ALL the most predominant type ( greater than 80%) occurring in childhood. Chronic leukaemias are malignancies in which the uncontrolled proliferating leukaemic cells are capable of maturation. Two subtypes are distinguished, chronic lymphocytic leukaemia (CLL) and chronic myeloid leukaemia (CML). Within these four groups, a considerable heterogeneity in biology and prognosis is seen, which currently is stratified along morphological features. This stratification bears as yet little value as to an understanding and prediction of the prognosis of a leukaemic patient and to rational therapy design.
However, recent molecular genetic studies of leukaemic patients have shown that a wide variety of chromosomal aberrations can be found with the various forms of leukaemia. One group consists of immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangements, comprising antigen-receptor gene rearrangements that go beyond the normal, physiological processes that are required to generate the diversity of the antigen receptor molecules which typify the lymphoid cell population. In one large group of IG and TCR rearrangements known to be associated with leukaemia, tumour specific antigen receptor molecules are expressed. Another group of aberrations comprise deletions of a whole gene or parts of a gene from a genome. As a result of the deletion, promotor regions normally belonging to the now deleted gene can exert control over another gene, resulting in aberrant transcription the gene. An example is the deletion of the coding regions of the SIL gene in T-cells, resulting in the transcription of the normally not expressed TAL1 gene in T-cells, resulting in ectopic expression of TAL-1 fusion protein. Yet another group comprises translocations of gene fragments between chromosomes, resulting in fusion genes that may well transcribe unique fusion proteins that contribute to the development of the malignancy. Well known examples are the translocations resulting in BCR-ABL fusion genes found in  greater than 95% of cases of CML and in 30% of cases of adult ALL and TEL-AML1 which is found in 25-30% of cases of childhood ALL. However, many more fusion genes, such as E2A-PBX1, ETO-AML1 and PML-RARxcex1 are known.
Chromosome aberrations can be detected by a wide array of techniques, various of which entail modern biomolecular technology. Traditional techniques such as cytogenetic analyses by conventional chromosomal banding techniques are, although highly precise, very labour intensive, require skilled personal and are thus expensive. Automated karyotyping is useful for some diagnostic applications, such as prenatal diagnosis, but is ineffective in analysing complex characteristics of malignancies. Furthermore, it is possible to detect increased activity of proteins, for example tyrosine-kinase activity (see WO 95/31545) in tumour specific cells. Above techniques require fresh cells, which are not always available. Other, more modern, techniques using Southern blotting or other nucleic acid hybridisation techniques or amplification techniques such as PCR, for detecting well-established chromosome aberrations for which suitable nucleic acid probes or primers are available. With these techniques, fresh or frozen cells can be used, and sometimes even older samples which have been stored appropriately (such as after formalin fixation), as long as the nucleic acid to be hybridised or amplified remains accessible and intact. However, even with the above modern technology, several disadvantages can be found that hamper the application of these diagnostic techniques in the rapid screening for chromosomal aberrations related to said malignancies.
Southern blotting lasts 3 to 4 weeks, which is much too slow to permit therapeutic intervention in malignancies, and allows only 10-15 kb of nucleic acid to be analysed per probe analysis.
PCR, although in essence well-suited for rapid and massive diagnostic testing or even screening, allows only 0.1 to 2 kb of nucleic acid (DNA or RNA) to be analysed per PCR analysis, which greatly hampers rapid screening of vast stretches of chromosomes and breakpoint cluster or fusion regions within the chromosomes or their gene-products. An additional disadvantage of PCR is its inherent sensibility to mismatched primers. Small, normal and physiological, alterations which can always be present in the nucleic acid sequence of the gene fragment complementary to the primer will make it impossible to operate the PCR with the wanted effect and may result in misdiagnosis and false-negative results. Especially false-negative results render a PCR-based diagnostic test, albeit very specific, not sensitive enough for reliable diagnosis, and it goes without saying that only a reliable diagnosis of malignancies can contribute to an understanding of the prognosis and the design of an adequate therapy.
Fluorescent in situ hybridisation techniques (FISH) are not so strongly dependent on the exact matching of nucleic acid sequences to get positive diagnostic results, but can only be employed for the detection of chromosomal DNA and not for the detection of the gene-products of the chromosomes. In general, FISH employs probe analyses with large, mainly unspecified, nucleic acid probes that hybridise, however often with varying stringency, with the genes or gene fragments located in the rearranged chromosome in the malignant cell. Using large probes of renders the FISH technique very sensitive. The binding of the probes is detected by subsequent detection of the probes with (often multiple) fluorochromes via microscopical observation of a population of cells obtained from the tested sample.
However, even the currently used FISH protocols have inherent disadvantages, mainly relating to the selection of nucleic acid probes employed in the current FISH protocols, often resulting in false-positive results in the diagnosis of chromosomal aberrations, resulting in diagnostic tests that are, albeit sensitive, not very specific, at least not specific enough to employ standard FISH techniques in massive or rapid diagnostic testing, let alone in automated testing or screening. A false-positive result necessitates cumbersome re-testing of patients, or even unsuspecting clients that have been submitted to routine screening protocols, and can greatly alarm these people.
Immunological detection of the fusion proteins resulting from chromosomal aberrations has, although widely tried, never been successful. This is caused mainly by the fact that it is hard to find immunological reagents that are exclusively reactive with tumour-specific proteins contrary to immunological detection of non-fusion proteins that are normally also produced by the body, albeit at a lower level (see for example Nagasaki et al., J.Imm. Methods 162, 235-245, 1993). Usually, such antibodies cross-react with normal cellular proteins. Only when specific fusion points are known, may it be possible to select specific immunological reagents that react exclusively with the tumour-specific protein, by selective binding to the fusion point epitope. However, the variation in fusion points is so large that specific immunological detection only works in a few occasions, often solely on a patient-by-patient basis.
Furthermore, the above diagnostic tests have the great inherent disadvantage that they require specialised and well equipped laboratories and trained and highly skilled personal. Furthermore, the above tests are only used in suspected cases of malignancies, and are not suitable for large scale screening of populations at risk for the presence of chromosomal aberrations. Large scale and preventive screening may lead to the early detection of malignancies, after which the often fatal course of a malignancy can be intercepted in an early phase of its development.
The present invention now provides a method to be used in diagnostic testing of biological samples such as blood samples, serum samples, samples of cells, tissue samples, bone marrow, biopsies, for chromosome aberrations. The invention provides a method to be used in diagnostic testing where both a high sensitivity as well as a high specificity is required. The invention provides a method that can optionally be performed in routine laboratories by personal with ordinary skills.
The present invention is characterized by a method to detect chromosomal aberrations in a biological sample via the exclusive detection of tumour-specific gene-product using at least two different probes directed against the tumour-specific gene-product originating from the chromosomal aberration. A surprising advantage of the invention is that the invention provides a method to detect chromosomal aberrations related to a wide array of types of cancer, for example, the invention provides a method to detect chromosomal aberrations related to leukaemia. The invention provides a method to detect tumour-specific gene products of various types of chromosomal aberrations, for example the invention provides a method to detect gene-products corresponding to the fused genes found in chromosomal deletions, inversions or translocations. As an example of the invention a method is provided to detect the Philadelphia chromosomal aberration found in leukaemias. The invention provides a method to detect tumour specific gene-products such as tumour-specific mRNA as well as tumour-specific protein. The probes used by the invention are optionally adjusted to the nature of the gene product, mRNA detection is provided by using at least two different nucleic acid probes, each being reactive with distinct sites on the gene-product. Tumour-specific protein detection is provided by using as probes at least two different binding-proteins, each being reactive with distinct sites on the gene product. As binding proteins, a wide array of proteins is known in the art, such as receptor molecules, polyclonal or monoclonal (synthetic) antibodies, binding peptides or xe2x80x98phagexe2x80x99 antibodies derived via phage display techniques, and so on. By using antibodies, the invention provides a method to detect chromosomal aberrations immunologically. As an example of the invention a method is provided whereby the tumour-specific gene-product is detected by a sepharose-Western blotting procedure. As a yet another example of the invention a method is provided whereby the tumour-specific gene-product is detected by dip-stick assay. However, other methods, whereby the tumour-specific gene product is detected by at least two different probes are also provided by the invention. For example, the invention provides a method whereby MRNA derived from a fused gene is detected by at least two nucleic acid probes, whereby at least one is directed against a mRNA fragment comprising the 5xe2x80x2 site of the tumour-specific mRNA, and at least one other one is directed against a mRNA fragment comprising the 3xe2x80x2 site of the tumour-specific mRNA, said fragments each corresponding to a non-tumour-specific mRNA. Furthermore, the invention provides a method whereby protein derived from a fused gene is detected by at least two binding proteins, whereby at least one is directed against a protein fragment comprising the amino-terminal fragment of the tumour-specific protein, and at least one other one is directed against a protein fragment comprising the carboxy-terminal fragment of the tumour-specific protein, said fragments each corresponding to a non-tumour-specific protein. As an example the invention provides a method to detect tumour-specific gene product whereby the amino-terminal protein fragment of the gene product corresponds to the ABL or BCR protein whereas the carboxy-terminal protein fragment corresponds to the BCR or ABL protein, respectively. With this example, probes are used that have similar antigen specificities as seen for antibodies 7C6, ER-FP1, Yae,8E9, G98-271.1.3, as shown in the experimental part. The invention provides a method using probes that can be labelled or conjugated with reporter molecules, such as biotin, digyoxigenin, enzymes such as peroxidase, alkaline phosphatase, or other reporter molecules known in the art. The invention further provides a diagnostic kit comprising all the means, such as (labelled) probes or reagents or substrate or instructions, necessary to carry out the method according to the invention. Methods or diagnostic kits provided by the invention are preferably used to detect chromosomal aberrations found with certain types of cancer, for example with leukaemia, be it in the detection of (residual) cancer in patients or the screening for cancer in larger populations as a whole.